Analysis and detection of chlamydial DNA

Elementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6.7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.     

Limited dispersal and an unexpected aggression pattern in a native supercolonial ant

Understanding how social groups function requires studies on how individuals move across the landscape and interact with each other. Ant supercolonies are extreme cooperative units that may consist of thousands of interconnected nests, and their individuals cooperate over large spatial scales. However, the inner structure of suggested supercolonial (or unicolonial) societies has rarely been extensively studied using both genetic and behavioral analyses. We describe a dense supercolony‐like aggregation of more than 1,300 nests of the ant Formica (Coptoformica) pressilabris. We performed aggression assays and found that, while aggression levels were generally low, there was some aggression within the assumed supercolony. The occurrence of aggression increased with distance from the focal nest, in accordance with the genetically viscous population structure we observe by using 10 DNA microsatellite markers. However, the aggressive interactions do not follow any clear pattern that would allow specifying colony borders within the area. The genetic data indicate limited gene flow within and away from the supercolony. Our results show that a Formica supercolony is not necessarily a single unit but can be a more fluid mosaic of aggressive and amicable interactions instead, highlighting the need to study internest interactions in detail when describing supercolonies.     

The association between the total antioxidant potential of plasma and the presence of coronary heart disease and renal dysfunction in patients with NIDDM

Oxidative stress may be an important pathogenetic factor in the development of diabetic vascular complications. The total antioxidative potential of plasma reflects the ability of an individual to resist oxidative stress. We measured the plasma total peroxyl radical-trapping potential (TRAP) and the concentrations of four plasma chain-breaking antioxidants in 81 patients with non-insulin-dependent diabetes mellitus (NIDDM) nine years after diagnosis and in 102 well-matched non-diabetic control subjects. The association between the total antioxidative potential and the presence of coronary heart disease (CHD) and diabetic kidney disease were also studied. There were no significant differences in plasma TRAP between NIDDM patients and control subjects (1250 ± 199 vs. 1224 ± 198 μM). Nor were there any significant differences in the concentrations of plasma uric acid, ascorbic acid, α-tocopherol, and protein thiols between NIDDM patients and control subjects. Patients with a low glomerular filtration rate and/or high urinary albumin excretion had elevated plasma uric acid. Plasma TRAP was not, however, associated with renal dysfunction. The plasma of NIDDM patients with CHD had a significantly higher value of unidentified antioxidative potential than that of patients without CHD. This relation was strongly dependent upon smoking. In conclusion, these data demonstrate that there are no major defects in the antioxidative potential of plasma caused by NIDDM per se. CHD and diabetic renal dysfunction were not associated with changes in plasma TRAP.     

Genetic Loci Associated with Alzheimer’s Disease and Cerebrospinal Fluid Biomarkers in a Finnish Case-Control Cohort

Objectives

To understand the relation between risk genes for Alzheimer’s disease (AD) and their influence on biomarkers for AD, we examined the association of AD in the Finnish cohort with single nucleotide polymorphisms (SNPs) from top AlzGene loci, genome-wide association studies (GWAS), and candidate gene studies; and tested the correlation between these SNPs and AD markers Aβ_1–42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF).

Methods

We tested 25 SNPs for genetic association with clinical AD in our cohort comprised of 890 AD patients and 701-age matched healthy controls using logistic regression. For the correlational study with biomarkers, we tested 36 SNPs in a subset of 222 AD patients with available CSF using mixed models. Statistical analyses were adjusted for age, gender and APOE status. False discovery rate for multiple testing was applied. All participants were from academic hospital and research institutions in Finland.

Results

APOE-ε4, CLU rs11136000, and MS4A4A rs2304933 correlated with significantly decreased Aβ_1–42 (corrected p<0.05). At an uncorrected p<0.05, PPP3R1 rs1868402 and MAPT rs2435211 were related with increased t-tau; while SORL1 rs73595277 and MAPT rs16940758, with increased p-tau. Only TOMM40 rs2075650 showed association with clinical AD after adjusting for APOE-ε4 (p = 0.007), but not after multiple test correction (p>0.05).

Conclusions

We provide evidence that APOE-ε4, CLU and MS4A4A, which have been identified in GWAS to be associated with AD, also significantly reduced CSF Aβ_1–42 in AD. None of the other AlzGene and GWAS loci showed significant effects on CSF tau. The effects of other SNPs on CSF biomarkers and clinical AD diagnosis did not reach statistical significance. Our findings suggest that APOE-ε4, CLU and MS4A4A influence both AD risk and CSF Aβ_1–42.     

Backbone NMR assignment of the internal interaction site of ALP

Earlier reports have shown that ALP has an internal interaction site. We were able to stablize the structure of this unfolded part to a great extent by aspartic acid, which allowed the backbone assignment. No secondary structure of the polypeptide was observed.     

Genetics of osteopontin in patients with chronic kidney disease: The German Chronic Kidney Disease study

Osteopontin (OPN), encoded by SPP1, is a phosphorylated glycoprotein predominantly synthesized in kidney tissue. Increased OPN mRNA and protein expression correlates with proteinuria, reduced creatinine clearance, and kidney fibrosis in animal models of kidney disease. But its genetic underpinnings are incompletely understood. We therefore conducted a genome-wide association study (GWAS) of OPN in a European chronic kidney disease (CKD) population. Using data from participants of the German Chronic Kidney Disease (GCKD) study (N = 4,897), a GWAS (minor allele frequency [MAF]≥1%) and aggregated variant testing (AVT, MAF<1%) of ELISA-quantified serum OPN, adjusted for age, sex, estimated glomerular filtration rate (eGFR), and urinary albumin-to-creatinine ratio (UACR) was conducted. In the project, GCKD participants had a mean age of 60 years (SD 12), median eGFR of 46 mL/min/1.73m² (p25: 37, p75: 57) and median UACR of 50 mg/g (p25: 9, p75: 383). GWAS revealed 3 loci (p<5.0E-08), two of which replicated in the population-based Young Finns Study (YFS) cohort (p<1.67E-03): rs10011284, upstream of SPP1 encoding the OPN protein and related to OPN production, and rs4253311, mapping into KLKB1 encoding prekallikrein (PK), which is processed to kallikrein (KAL) implicated through the kinin-kallikrein system (KKS) in blood pressure control, inflammation, blood coagulation, cancer, and cardiovascular disease. The SPP1 gene was also identified by AVT (p = 2.5E-8), comprising 7 splice-site and missense variants. Among others, downstream analyses revealed colocalization of the OPN association signal at SPP1 with expression in pancreas tissue, and at KLKB1 with various plasma proteins in trans, and with phenotypes (bone disorder, deep venous thrombosis) in human tissue. In summary, this GWAS of OPN levels revealed two replicated associations. The KLKB1 locus connects the function of OPN with PK, suggestive of possible further post-translation processing of OPN. Further studies are needed to elucidate the complex role of OPN within human (patho)physiology.     

Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs.     

Fine mapping of the Schnyder’s crystalline corneal dystrophy locus

Schnyders crystalline corneal dystrophy (SCCD) is a rare autosomal dominant eye disease with a spectrum of clinical manifestations that may include bilateral corneal clouding, arcus lipoides, and anterior corneal crystalline cholesterol deposition. We have previously performed a genome-wide linkage analysis on two large Swede-Finn families and mapped the SCCD locus to a 16-cM interval between markers D1S2633 and D1S228 on chromosome 1p36. We have collected 11 additional families from Finland, Germany, Turkey, and USA to narrow the critical region for SCCD. Here, we have used haplotype analysis with densely spaced microsatellite markers in a total of 13 families to refine the candidate interval. A common disease haplotype was observed among the four Swede-Finn families indicating the presence of a founder effect. Recombination results from all 13 families refined the SCCD locus to 2.32 Mbp between markers D1S1160 and D1S1635. Within this interval, identity-by-state was present in all 13 families for two markers D1S244 and D1S3153, further refining the candidate region to 1.58 Mbp.     

Circulating oxidized low-density lipoprotein and common carotid artery intima-media thickness in a random sample of middle-aged men

Circulating oxidized low-density lipoprotein (oxLDL) has been suggested to play an important role in atherosclerosis development. According to previous observations, oxLDL correlates with clinically manifest coronary and carotid artery disease. We investigated the association between the oxLDL concentration measured directly in plasma and common carotid artery intima-media thickness (IMT) in a population-based, case-control study in middle-aged men from Southern Finland. oxLDL was determined in 214 men by a commercially available sandwich ELISA test (Mercodia). Carotid artery IMT was measured at 12 standardized segments by B-mode ultrasonography (at the near and far wall of the left and right common carotid arteries, bifurcations and internal carotid arteries), and the overall mean maximum IMT (MMaxIMT) was calculated. The MMaxIMT of the carotid arteries was significantly associated with circulating oxLDL (r_s=0.16, p=0.018). In a stepwise multiple regression model with MMaxIMT as dependent variable and systolic blood pressure, smoking, oxLDL, HDL cholesterol and apolipoprotein B as covariates, systolic blood pressure (=0.22, p<0.001), oxLDL (=0.15, p=0.022) and smoking (=0.17, p=0.014) showed an independent association with IMT (R²=0.10, p<0.001). Our results show that oxLDL measured directly from plasma is independently associated with subclinical carotid artery atherosclerosis in middle-aged men.